Download Drug resistance in fungi – an emerging problem

Drug resistance in fungi – an emerging problem
Arunaloke Chakrabarti*
Abstract
Over the past quarter of a century, invasive fungal infections have emerged as an important cause of
morbidity and mortality in immunocompromised patients. Although several new antifungal drugs have
been licensed in recent years, antifungal drug resistance is becoming a major concern during treatment of
such patients. The resistance may be intrinsic, acquired or clinical. The understanding of the mechanism
of resistance and clinical impact is important while planning treatment strategies. Four altered gene
expression pathways have been identified in azole resistance. The mechanism of resistance in polyene and
echinocandins is still not clearly understood. Recent studies have revealed that molecular chaperone heat
shock protein (Hsp90) can alter the relationship between genotype and phenotype leading to a profound
impact on antifungal drug resistance. Though definite progress has been made to correlate standardized
in vitro antifungal susceptibility testing with prediction of treatment outcome, limitations still exist due to
time required for testing and understanding the factors leading to clinical resistance. Overall, the level of
resistance to antifungal agents is still relatively low, but there is a possibility of antifungal resistance
becoming a crucial determinant of outcome following antifungal therapy in future.
Introduction
Medical progress has led to an expanding
population of susceptible hosts with impaired
immunological defenses against infection in
the community and hospitals. These
populations are at heightened risk for many
opportunistic fungal diseases including
candidiasis,
aspergillosis,
mucormycosis
(zygomycosis),
cryptococcosis,
pneumocystosis.
Traditionally
Candida
and
Aspergillus species accounted for the majority
of infections. Candidemia is the fourth leading
cause of blood-stream infections and carries
35-55% mortality1. The incidence of mould
infections has also increased in the recent
past, especially infectious caused by
Aspergillus spp. where the mortality rate
crosses 50% in such patients2. Mucormycosis
is a threat in uncontrolled diabetes in
developing countries like India3. In this
* Department of Medical Microbiology, Postgraduate
Institute of Medical Education and Research,
Chandigarh
Regional Health Forum – Volume 15, Number 1, 2011
scenario,
contemporary
epidemiological
trends also indicate a certain shift of the fungal
pathogen towards resistant species among
those common two genera, Candida and
Aspergillus, and emergence of the previously
uncommon fungi that are particularly difficult
to manage4,5. These include C. glabrata and
C. krusei in yeast with their reduced drug
susceptibility, and among the mould fungi,
these include the non-fumigatus Aspergillus
spp. like Aspergillus terreus, zygomycetes and
Fusarium spp. Selective pressure due to
increased use of antifungal prophylaxis in
high-risk patients has been suggested as a
contributory factor for this shift and emergence
of uncommon mould6.
Antifungal drugs and the problem of
resistance
For a long time amphotericin B deoxycholate
and 5 fluorocytosine were the only therapeutic
options for invasive fungal infections. The first
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therapeutic alternatives began to emerge with
the introduction of fluconazole and
itraconazole in the late 1980s. Expansion in
antifungal research in the last two decades has
led to the development of lipid formulations of
amphotericin B (amphotericin B colloidal
dispersion, amphotericin B lipid complex, and
liposomal amphotericin B), a secondgeneration
broad
spectrum
triazoles
(voriconazole, posaconazole) and an entirely
new class of antifungal agents, the
echinocandins (caspofungin, anidulafungin
and micafungin). A few of the new antifungal
agents are under clinical trial (Table 1).
Despite the increase in the spectrum of
antifungal agents now available, the choice of
suitable antifungal agents remains relatively
limited due to the emergence of comparatively
more resistant fungal species, slow
mycological
diagnosis,
variable
drug
bioavailability
in
immunocompromised
patients, toxicity of antifungal agents, lack of
either oral or intravenous preparations, drug
interaction, and most importantly due to
development of resistance and breakthrough
infections7, 8.
Unfortunately, the increased use of
triazoles in prophylactic and empiric antifungal
therapy in high-risk patients has led to selective
pressure towards drug-resistant Candida and
Aspergillus species9. It has resulted in infection
either through the inherently resistant fungi
(primary resistance) or through the resistant
subpopulation of the normally susceptible
fungi (secondary resistance). Fortunately,
development of acquired resistance in fungi is
not a “fast-track” event as in bacteria or
viruses, except in the event of nearly one third
patients with advanced AIDS harbouring
fluconazole-resistant C. albicans in their oral
cavity10. As no known mechanism of horizontal
resistance gene transfer was known in fungi, it
was believed that exceptionally large number
of viable fungi when exposed to high levels of
antifungals in the oropharyngeal candidiasis
might become resistant to antifungal agents11.
The episode of rapid emergence of antifungal
resistance ended with the advent of effective
antiretroviral therapy in patients with AIDS.
However, there is no scope for complacency
as recently in a genome-wide analysis of three
Fusarium species, it was shown experimentally
that complete chromosomes could be
transferred between different fungal strains12.
Prior to this it was believed that fungi were
generally confined to vertical gene transfer, a
slower type of genetic change based on
mutation, recombination and the effect of
Table 1: Currently available and “under trial” antifungal agents
Compound
Currently available
Under clinical trial
Polyenes
Amphotericin B deoxycholate and
lipid formulations (amphotericin B
lipid complex, amphotericin B colloid
dispersion, liposomal amphotericin B)
Fluorinated pyrimidine
5 fluorocytosine
Triazoles
Fluconazole, itraconazole,
voriconazole, posaconazole
Isavuconazole,
ravuconazole,
albconazole
Ergosterol biosynthesis - 14α
demethylase
Echinocandins
Caspofungin, anidulafungin,
micafungin
Aminocandins
1-3-β-d glucan synthesis in cell wall
Allylamines
Terbinafine
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Liposomal nystatin
Target site
Ergosterol in cell membrane
DNA, RNA synthesis
Ergosterol biosynthesis – squalene
epioxidase
Regional Health Forum – Volume 15, Number 1, 2011
selection. This new understanding of fungal
genetics would help researchers understand
the types of fungi that are most likely to
develop resistance to antifungal agents.
The overall resistance in Candida spp. to
fluconazole and voriconazole is considered to
be around 3-6% and level of resistance has
remained constant over a decade13. However,
a recent report from India revealed panazole
resistance in ~10% of Candida species14.
Triazole resistance in A. fumigatus is
increasingly being recognized and up to 6% of
clinical isolates were found to be resistant to
triazole in the United Kingdom and the
Netherlands15, 16. In contrast to azoles,
echinocandin resistance does not seem to be
the major cause of concern, as global
surveillance studies indicate that there has not
been any significant epidemiological shift in
the susceptibility of Candida spp. isolates to
echinocandins17. However, since 2005 there
have been multiple case reports of
breakthrough infections after echinocandin
therapy in patients with AIDS or acute myeloid
leukaemia9, 18. The prevalence of flucytosine
resistance in yeast remains low (<2%). But the
speed at which yeast can develop resistance to
flucytosine has prompted clinicians to use the
compound in combination with mainly
amphotericin B19. Overall, though the
incidence of antifungal resistance is low, it
remains a serious problem in the management
of high-risk patients. Recently, concern has
been expressed on the possibility of induction
of resistance in opportunistic fungi in the
environment as azole fungicides are used in
agriculture20.
Mechanism of antifungal resistance
The mechanism of drug resistance in
microorganisms traditionally takes the path of
either identifying a cellular determinant that
prevents entry of the drug or removes the drug
from the cell or inactivates the drug or prevents
the drug from inhibiting the target of various
combinations of the above-mentioned
pathways. In fungi, mutation in gene encoding
target proteins, up-regulations of expression of
multidrug efflux pumps and drug target
themselves, altering the stoichiometry of the
inhibitor target ratio in favour of fungus are
possible mechanisms19. However, no fungus
has yet been shown to have the ability to
degrade an antifungal agent like betalactamase in bacteria. Multidrug resistance,
called pleiotrophic drug resistance (POR) in
Saccharomyces cerevisiae is possibly an
ancient model for multidrug resistance that
operates in pathogenic fungi through the efflux
pump21. Therefore, most studies on the
antifungal drug resistance mechanism have
targeted the efflux pump mechanism.
Inhibition of the pump over-expression or drug
pump activity may transform a fungistatic drug
like azole into a fungicidal drug. In C. albicans
a unique mechanism of gene amplification
leading to azole resistance has been identified.
The mechanism involves formation of
aneuploidy or isochromorsome, in which the
chromosome arm bearing both transcription
factor (regulating ABC transporter) and target
of the azoles Erg11 is duplicated22. The
different genetic alterations and mechanism of
resistance in Candida spp. and Aspergillus
spp. are summarized in Table 2.
Table 2: Genetic mechanism of resistance in Candida and Aspergillus (modified from reference 9)
Antifungal Azoles
Candida
Aspergillus
-Decreased drug concentration (efflux pumps)
↑CDR gene of ATP binding cassette (for all azoles)
↑MDR gene of major facilitator class (for fluconazole)
Mdr1, Mdr3, Mdr4
C. albicans CDR1, CDR2, MDR1
C. glabrata CgCDR1, PDH1, Snq2
C. dubliniensis cdCDR1, CdMDR1
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Antifungal Azoles
Candida
Aspergillus
-Target cell alteration
-Mutation of ERG11
-Decrease affinity ERG11p (intrinsic resistance to fluconazole in C. krusei
isolates)
-↑ERG11p
-Cyp51A (mutation at codon 220
develops resistance to all azoles,
mutation at codon 54 develops crossresistance to itraconazole and
fluconazole)
-↑Cyp51A
-Bypass pathway
-Mutation of ERG3 (prevent formation of toxic products from 14-α methyl
fectosterol)
-Chromosomal aneuploidy or isochromosome
C. albicans – chromosome 5
Polyenes
-Target site alteration
-Mutation of ERG3 (accumulation of other sterols)
-Alteration of drug : target ration by any mechanism
Echinocandins
-Target site alteration
-Point mutation mostly at Ser 645 of Fks1
Fks1
C. albicans Fks1
C. glabrata Fks1, Fks2
-Activation of salvage or compensatory pathway for chitin synthesis (PKC
cell integrity pathway)
From the evolutionary perspective, none
of the mechanisms acts alone. Phenotypic
resistance depends on the genetic variation
occurring in a particular genome. However,
the development of resistance is often
accompanied by a deleterious effect of
mutation on the fitness of fungi in the absence
of the drug. Compensatory mutation may
mitigate this effect and enhance fitness23.
Hsp90 is known to play an important role in
remodelling
the
relationship
between
phenotype and genotype in distant species. In
antifungal drug resistance its role has been
emphasized recently23, 24. Hsp 90 acts as a
100
capacitor for accumulation of genetic
variation. When its function is compromised by
genetic alteration, pharmacological inhibitors
or environmental stress, genetic variations are
revealed, which lead to alteration of the
relationship between genotype and phenotype.
Hsp90 acts through calcineurin. Any inhibitor
of Hsp90 or calcineurin would possibly act
synergistically with the antifungal agent.
Antifungal drug susceptibility testing
Both the Clinical Laboratory Standard Institute
(CLSI), United States of America, and the
Regional Health Forum – Volume 15, Number 1, 2011
European Committee on Antimicrobial
Susceptibility Testing (EUCAST) have published
approved protocol antifungal susceptibility
testing either by broth microdilution or disc
diffusion assay. Drug threshold levels for
in vitro growth inhibition yield a minimum
inhibitory concentration (MIC). The CLSI has
recommended antifungal MIC breakpoints to
separate susceptible and resistant population
for azoles and echinocandins by analysing the
in vitro susceptibility data, in vitro outcome
and
pharmacokinetics/pharmacodynamic
studies6, 9, 25. However, EUCAST defined the
breakpoint derived from MIC as the
Epidemiological Cut-off Value (ECV) to avoid
confusion with clinical breakpoints. The
EUCAST uses ECV “as the most sensitive
measure of resistance development — for
measuring resistance development in hospitals
and the community, for measuring the effect of
interventions and for developing strategies to
counteract further resistance development26”.
The breakpoint derived through MIC tends to
be lower than the clinical breakpoint, as this
procedure is independent of dosage regimens.
In contrast, the clinical breakpoint is based on
distribution of MIC, pharmacokinetics of the
antimicrobial agent, and the clinical outcome.
Therefore, “the clinical breakpoint should be
used in every day clinical laboratory work to
provide evidence for rational therapy in the
patient”26. While correlating the therapeutic
outcome in multiple studies with in vitro
antifungal susceptibility testing data, especially
the combination of Candida species and azole
antifungal agents, a pattern of “90-60” rule
emerged like in bacteria: infections due to
susceptible isolates respond to therapy ~90%
of the time, whereas infections due to resistant
isolate respond ~60 % of the time27.
However, in spite of all these studies and
recommended
standards,
antifungal
susceptibility
testing
rarely
influences
management protocol in an individual patient,
as it takes 48-72 hours after isolation of the
fungus. Therefore, there is a need for a more
rapid test procedure or “real time” antifungal
susceptibility testing for clinicians.
Regional Health Forum – Volume 15, Number 1, 2011
Cross-resistance among antifungal agents
Cross-resistance among azoles is expected as
the target of action on fungi is similar. In HIVpositive patients a high level of crossresistance to itraconazole was observed in
fluconazole–resistant
C.
glabrata
and
C. tropicalis compared with C. albicans and
C. krusei isolates28. Cross-resistance in
C. glabrata strains was due to increased
expression of CgCDR1, CgCDR2 genes and
CDR efflux pumps. Though voriconazole also
has cross-resistance with other azoles, the rate
is low, and after performing in vitro
susceptibility testing it may be used in patients
who have previously been exposed to
fluconazole or itraconazole6. However, in a
study conducted in India, high cross-resistance
to fluconazole, itraconazole and voriconazole
was reported in C. albicans and C. tropicalis
blood isolates, though the mechanism of
resistance was not studied.14 Cross-resistance
has been observed among the three
echinocandins.17, 18 Cross-resistance should
not be expected between the echinocandin
class of drugs and either the polyene or
azoles, as the sites of action are different.
Clinical antifungal resistance
Non-specific symptoms and signs of invasive
fungal infections present difficulties in early
diagnosis; delay in diagnosis is the major
cause of treatment failure28. Even in empiric
and targeted therapy the success rate ranged
from 32% to 74%19. The major causes of
treatment failure have been summarized by
Kanafani and Perfect19: (i) incorrect diagnosis
of specific fungal disease including immune
reconstitution inflammatory syndrome (IRIS) in
patients with AIDS after antiretroviral therapy;
(ii) failure of antifungal agents to overcome the
state of severe immune deficiency in such
patients; (iii) more virulent infection such as
Cryptococcus gattii infections; (iv) toxicities of
antifungal agents (nephrotoxicity in polyene
and hepatitis in azoles; (v) poor penetration of
antifungal agents at certain sites of fungal
101
infections such as the central nervous system
or the necrotic tissue with poor blood supply;
(vi) reduced blood concentration of the
antifungal agent due to drug interaction,
especially during voriconazole therapy;
(vii) suboptimal duration of the antifungal
therapy; and (viii) the underlying disease as the
main barometer of clinical success and failure
in antifungal therapy.
Like bacteria, fungi also produce a biofilm
in vitro. It is well known that the biofilm is an
important obstruction in antibacterial therapy.
In fungal infections similar studies have been
conducted. Enhanced extracellular matrix
especially beta glucan synthesis during biofilm
growth has been shown to prevent penetration
of antifungal agents such as azole and
polyene29. It is believed that the echinocandins
and lipid formulations of amphotericin B can
penetrate biofilm better than amphotericin
B deoxycholate and azoles19. The clinical trials
also indicate the importance of the biofilm.
Numerous clinical trials on candidemia have
demonstrated that the treatment failure and
mortality are high in patients who are on
catheters for long periods.30
Conclusion
With increase in the incidence and spectrum of
invasive fungal infections, antifungal drug
resistance has become an important
consideration in the management of patients.
Though unlike bacteria, the level of resistance
to antifungal agents is relatively low due to the
possible absence of drug-resistant plasmid or
transposons in fungi, the recently conducted
experiment of horizontal gene transfer in
pathogenic Fusarium species shows that there
is no scope of complacency. The emergence
of intrinsically resistant fungal species as a
human pathogen is compounding the
challenge of planning treatment strategies.
Beyond these confounding factors, the
conditions leading to clinical resistance should
be kept in mind while managing invasive
fungal infections in immunocompromised
patients.
Acknowledgement
The author acknowledges the help of
M. Manpreet Dhaliwal for organizing the
references.
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